1. Field of the Invention
This invention relates generally to mechanisms of gene expression in plants and more specifically to regulation of expression of genes in plants in a "tissue-preferred" manner. A method for isolation of transcriptional regulatory elements that contribute to tissue-preferred gene expression is disclosed. Transcriptional regulatory elements isolated using the methods of this invention are demonstrated to direct tissue-preferred gene expression of genes within certain tissues of a plant. DNA molecules representing tissue-preferred transcriptional regulatory elements and vectors comprising said DNA molecules are demonstrated. Said transcriptional regulatory unit will ultimately be utilized for driving tissue-preferred expression of at least one gene that confers a selective advantage upon a plant.
2. Description of the Related Art
Gene expression encompasses a number of steps from the DNA template to the final protein or protein product. Initiation of transcription of a gene is generally understood to be the predominant controlling factor in determining expression of a gene. The transcriptional controls are generally relegated to relatively short sequence elements embedded in the 5'-flanking or upstream region of the transcribed gene to which DNA-binding proteins may interact. These DNA sequence elements serve to promote the formation of transcriptional complexes and eventually initiate gene expression processes. A number of labs have identified promoter elements and corresponding DNA-binding proteins that are limited to specific tissues within the plant (Weising, et al Z. Naturforsch C46:1; Oeda, et al EMBO J. 10:1793; Takatsuji, et al EMBO J 11:241; Yanagisawa, et al Plant Mol. Biol. 19:545; Zhou, et al J. Biol. Chem. 267:23515; Consonni, et al Plant J. 3:335; Foley, et al Plant J. 3:669; Matsuoka, et al Proc. Natl. Acad. Sci. 90:9586). It is likely that a large number of DNA-binding factors will be limited to specific tissues, environmental conditions or developmental stages. It is considered important by those skilled in the art to develop transcriptional regulatory units that restrict gene expression to certain tissues of a plant. The ability to drive tissue-specific gene expression in plants is considered to be of agronomic importance to those skilled in the art.
Controlling the expression of agronomic genes in transgenic plants is considered by those skilled in the art to provide several advantages over generalized or constitutive expression. The ability to control gene expression may be utilized to exclude expression in germline tissues thus avoiding certain regulatory and commercial issues. It can also provide a pest refugia in the case of insect and disease resistance, a preferred pest management strategy (Norton, G., Agricultural Development Paths and Pest Management--A Pragmatic View of Sustainability, in Crop Protection and Sustainable Agriculture, 1993, p. 100-115), and it can reduce potential yield loss by limiting expression of some pernicious, yet useful agronomic genes to only specific tissues. Further advantages of utilizing promoters that function in a tissue-preferred manner include reduced resource drain on the plant in making a gene product constitutively, as well as localization or compartmentalization of gene expression in cases where the gene product must to be restricted to, or from, a certain tissue. Said gene products may include general cellular inhibitors such as RNases or other cytotoxins. As an example, Mariani, et al (Nature 347:737, 1990) demonstrated anther-specific gene expression of suc inhibitor genes for use in male sterility systems, since expression in regions other than the anther in a plant would be toxic. There is a need in the art for novel transcriptional regulatory elements capable of driving tissue-preferred gene expression in plants. It is considered important by those skilled in the art to continue to provide tissue-preferred transcription units capable of driving expression of genes that may confer a selective advantage to a plant.
It is also considered important to those skilled in the art to develop transcriptional regulatory units that direct gene expression selectively to root tissue. Root-preferred gene expression will provide several advantages to a plant including but not limited to alteration of the growth rate or function of the root tissue, resistance to root-preferred pathogens, pests, herbicides or adverse weather conditions as well as broadening the range of soils or environments in which said plant may thrive. Root-preferred gene expression would also provide a mechanism by which root morphology and metabolism may be altered to improve yield (i.e., direct expression of transporter proteins). Further advantages to root-preferred gene expression include the production of useful proteins in an industrial setting. Light-sensitive proteins may be synthesized in root tissue such that said proteins are not exposed to light.
A promoter element or elements that specifically confer root-preferred expression has not been described. Short elements that may contribute to root-preferred expression have been disclosed; however, identification of the specific sequences responsible for root-specific gene expression have not been reported (Lam, et al. 1989. Proc. Natl. Acad. Sci. USA 86:7890). Importantly, most of the disclosed elements are derived from dicots not from monocots (as was this invention). Based on what is known by those skilled in the art, it is unlikely that a dicot promoter sequence will function properly in a monocot.
Approaches that may be utilized to isolate tissue-specific transcriptional control elements include differential or subtractive cDNA cloning followed by cloning of the genomic 5'-flanking sequence comprising the promoter elements responsible for tissue-preferred gene expression. A recently developed technique, differential display analysis (Liang, et al Science 257:967; Bauer, et al Nuc. Acids Res. 21:4272), involves PCR-based identification of differentially expressed genes as partial cDNAs. A major drawback to the techniques taught in the prior art is that cloning of full length cDNA and genomic sequences as well as mapping of the identified promoter from genomic clones may be required. These techniques potentially require a multiple-year project with minimal returns on investment. The present invention, which is a subject of this application, addresses these issues by providing a methodology that allows for rapid isolation, identification and utilization of DNA elements that drive tissue-preferred gene expression.
This invention teaches a process of enrichment of novel DNA sequences that interact with tissue-specific DNA-binding proteins. DNA sequences so isolated may then be utilized to construct expression vectors useful in driving tissue-specific gene expression within transgenic tissues or organisms. A random oligonucleotide library (ROL) is designed and applied to a crude mixture of nuclear proteins from the target tissue (e.g. root) immobilized on a filter (designated as a Southwestern assay). The bound ROL are eluted and applied to an immobilized, crude mixture of nuclear proteins from the non-target tissue (e.g. leaf). Non-bound ROL are then PCR-amplified and the entire cycle repeated to further enrich for DNA sequences that bind to nuclear proteins from the target tissue (e.g. root). The use of ROL's binding directly to crude nuclear extracts in a subtractive enrichment process for generating a library of tissue (or developmental or environmental state)-specific promoter elements has not been described.
As demonstrated by Oliphant, et al. (Mol. Cell. Biol. 9:2944), ROL's have been utilized to identify alternative DNA recognition sequences of DNA-binding proteins. Variations of this technique have also been used to identify alternative sequence recognition of specific DNA-binding factors (Norby, et al Nuc. Acids Res. 20:6317; Catron, et al Mol. Cell. Biol. 13:2354; Ko, et al Mol. Cell. Bill. 13:4011; Shiraishi, et al Plant J. 4:385; Huang, et al Nuc. Acids Res. 21:4769). The technique of applying crude nuclear extracts to an ROL was successfully utilized to identify several oligonucleotides that bind to retinoblastoma tumor-suppressor gene products (Ouellette, et al. Oncogene 7:1075-1081). In each of the above-described techniques, however, a purified DNA-binding factor or antibody specific for a DNA binding factor is required. This invention teaches the isolation of tissue-specific oligonucleotides from a pool of random sequences using a heterogeneous mix (crude extract) of nuclear proteins without the need for either a purified protein or an antibody of any sort. The enriched collection of oligonucleotides are then isolated, cloned into expression vectors, and tested for the ability to drive tissue-specific gene expression. Importantly, the invention of the present application provides a significant methodological advancement over previously described techniques and is applicable to any plant or non-plant tissue that is amenable to nuclear extract preparation.